Methods and compositions for preparing surfactant protein d (sp-d)

Abstract

Some embodiments of the methods and compositions provided herein relate to the preparation surfactant protein-D (SP-D). Some embodiments include the expression of human SP-D in certain cell lines, and the purification of human SP-D from such cell lines. Some embodiments include the preparation of certain oligomeric forms of human SP-D.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority to U.S. Prov. App. No. 62 / 614774 filed Jan. 8, 2018 entitled “METHODS AND COMPOSITIONS FOR PREPARING SURFACTANT PROTEIN D (SP-D)”, and to U.S. Prov. App. No. 62 / 554777 filed Sep. 6, 2017 entitled “METHODS AND COMPOSITIONS FOR PREPARING SURFACTANT PROTEIN D (SP-D)”, which are each incorporated by reference in its entirety.REFERENCE TO SEQUENCE LISTING[0002]The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled AIRWY009ASEQ, created Aug. 31, 2018, which is approximately 13 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entiretyFIELD OF THE INVENTION[0003]Some embodiments of the methods and compositions provided herein relate to the preparation of surfactant protein-D (SP-D). Some embodiments include the expression of human SP-D in certain cell lines,...

AI Technical Summary

Benefits of technology

This patent is about a way to increase the amount of a specific protein called SP-D by using a chemical called methotrexate. This chemical can inhibit the growth of certain cells and is used to treat autoimmune diseases. The researchers found that by adding more methotrexate to the cells, they could increase the production of the SP-D protein. This may have technical uses in treating cancer or other diseases where the protein is low or inactive.

Problems solved by technology

However, treatment becomes more difficult if the lungs are infected or if there are inflammatory or oxidative complications, because current surfactant preparations lack surfactant protein D (SP-D).

The low SP-D yields and variable oligomerization states make it difficult to use natural sources for the production of pharmaceutical SP-D (Strong P, et al., J Immunol Methods (1998) 220:139-49).

However, it is challenging to express recombinant human SP-D (rhSP-D) to levels sufficient for a commercial campaign in commonly used mammalian cell lines because the protein is not synthesized efficiently and yields are typically <2 mg of purified protein per liter.

Although yields tend to be higher in non-mammalian systems, expression of only a truncated variant of SP-D has been attempted in systems such as yeast or bacteria which have the disadvantage of either not producing the glycosylated form of the protein, or not producing the protein with a human glycosylation pattern (Salgado D, et al., Front Immunol (2014) 5:623, doi: 10.3389 / fimmu.2014.00623).

Furthermore, it has not been possible to date to control the variability in oligomerization states seen with recombinant and natural human SP-D.

Unless the expression system can reproducibly produce rhSP-D with consistently stably levels of the higher-order multimerization states observed in natural SP-D, there is a potential for reduced efficacy of such preparations.

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